This disclosure resides in the fields of cellular engineering and drug discovery.
Much of the research activity of pharmaceutical companies in years past focused on the incremental improvement of existing drugs. These efforts involved repetitive rounds of compound modification and biological testing and have resulted in a large percentage of the available drugs directed to similar targets.
Approximately a dozen years ago, the emphasis of pharmaceutical research activities began shifting toward the purposeful discovery of novel chemical classes and novel molecular targets. This change in emphasis, and timely technological breakthroughs (e.g., molecular biology, laboratory automation, combinatorial chemistry) gave birth to high throughput screening, or HTS, which is now widespread throughout the biopharmaceutical industry.
High throughput screening involves several steps: creating an assay that is predictive of a particular physiological response; automating the assay so that it can be reproducibly performed a large number of times; and, sequentially testing samples from a chemical library to identify chemical structures able to xe2x80x9chitxe2x80x9d the assay, suggesting that such structures might be capable of provoking the intended physiological response. Hits from the high throughput screen are followed up in a variety of secondary assays to eliminate artifactual results, particularly toxic compounds.
A high throughput screen could involve the testing of 200,000 compound samples or more, therefore requiring the use of lab robots. Examples of samples tested in such an assay include pure compounds saved in compound archives (e.g., certain pharmaceutical companies have chemical libraries that have been generated through decades of medicinal chemistry effort), samples purchased from academic sources, natural product extracts and libraries purposefully created for high throughput screening such as combinatorial libraries.
The assays used in high throughput screens are intended to detect the presence of chemical samples possessing specific biological or biochemical properties. These properties are chosen to identify compounds with the potential to elicit a specific biological response when applied in vivo. High throughput screens typically identify drug candidates rather than the agents that will ultimately be used as drugs. A compound of a certain chemical class found to have some level of desired biological property in a high throughput assay can then be the basis for synthesis of derivative compounds by medicinal chemists.
The assays fall into two broad categories: biochemical assays and cell-based assays. Biochemical assays utilize pure or semi-pure components outside of a cellular environment. Enzyme assays and receptor binding assays are typical examples of biochemical assays. Cell-based assays utilize intact cells in culture. Examples of such assays include luciferase reporter gene assays and calcium flux assays.
Biochemical assays are usually easier to perform and are generally less prone to artifacts than conventional cell-based assays. Compounds identified as xe2x80x9cactivexe2x80x9d in a biochemical assay typically function according to a desired mechanism, decreasing the amount of follow-up experimentation required to confirm a compound""s status as a xe2x80x9chit.xe2x80x9d A major disadvantage of biochemical assays, however, is the lack of biological context. Compound xe2x80x9chitsxe2x80x9d from biochemical screens do not have to traverse a plasma membrane or other structures to reach and affect the target protein. Consequently, biochemical assays tend to be far less predictive of a compound""s activity in an animal than cell-based assays.
Cell-based assays preserve much of the biological context of a molecular target. Compounds that cannot pass through the plasma membrane or that are toxic to the cell are not pursued. This context, however, adds complexity to the assay. Therefore conventional cell-based assays are much more prone to artifact or false positive results than are biochemical assays. Compounds that trigger complex toxic reactions or trigger apoptosis are particularly troublesome. Much of the labor devoted to conventional cell-based high throughput screening is directed to follow-up assays that detect false hits or hits that work by undesirable mechanisms.
If false positive or artifactual hits could be rapidly identified and eliminated, the ease and efficiency of biochemical assays could be approached in cell-based assays, while preserving the biological context. The result would be an assay with optimum throughput and optimum predictability of biological function. In short, a more efficient process for the discovery of new pharmaceuticals would be produced.
In one aspect, methods of screening a compound for interaction with a molecular target are provided. In certain embodiments, the method involves the following steps: (a) contacting a first cell with the compound; (b) determining a first value of a property of the first cell, the property being responsive to the cell being contacted with the compound; (c) contacting a second cell with the compound, wherein the second cell comprises an exogenous zinc finger protein that directly or indirectly modulates expression of the molecular target; (d) determining a second value of the property in the second cell. A difference between the value of the cell property in the first cell and the cell property in the second cell provides an indication of an interaction between the compound and the molecular target. The zinc finger protein preferably modulates expression of the molecular target itself, but, in some embodiments, may indirectly modulate expression of the molecular target for example, by modulating expression of a protein that then modulates and/or affects the molecular target. Therefore, using these screening methods, one can, for example, test a compound for its capacity to transduce a signal through the molecular target or its capacity to block transduction of a signal through the molecular target.
In certain embodiments, the first and second cells are substantially identical with the exception that the second cell contains an exogenous zinc finger protein and/or sequences encoding an exogenous zinc finger protein. In certain embodiments, there may be further genetic (and/or phenotypic) differences between the first and second cells.
In any of the methods described herein, the zinc finger protein can be a component of a fusion molecule, for example a fusion of a zinc finger protein and a functional domain. The functional domain may be, for example a repression domain such as KRAB, MBD-2B, v-ErbA, MBD3, unliganded TR, and members of the DNMT family; an activation domain such as VP16, the p65 subunit of NF-kappa B, ligand-bound TR, and VP64; an insulator domain; a chromatin remodeling protein or component of a chromatin remodeling complex; and/or a methyl binding domain. According to the methods, the zinc finger protein either activates or inhibits the expression of the target. The zinc finger protein (or fusion) can activate expression, for example, such that the expression level in the second cell is more than 125% or 175% of the expression level in the first cell. The zinc finger protein can inhibit expression, for example, such that the expression level in the second cell is less than 95%, 75%, 50%, 25% or 5% of the expression level in the first cell.
In certain embodiments, the molecular target is a protein. However, a molecular target is any molecule whose expression can be modulated by a zinc finger protein, for example, RNA, carbohydrate and/or lipid.
In any of the method described herein, the zinc finger protein (or fusion molecule) can be provided as a protein or as a polynucleotide encoding the protein or fusion molecule. Thus, according to the methods, the zinc finger protein is either expressed in, or added to, the second cell. In certain embodiments in which the zinc finger protein is provided as a polynucleotide, expression of the zinc finger protein can be inducible, for example using an inducible transcription control element (e.g., promoter) operably linked to the sequence encoding the zinc finger protein. In these embodiments, the first and second cells may both contain a polynucleotide encoding a zinc finger protein but expression is induced in only one of the two matched cells. Furthermore, the first and/or second cells may contain more than one exogenous zinc finger protein or fusion molecule (or polynucleotide encoding same).
In other aspects, the first and/or second cells used in the screening methods can also comprise a reporter (e.g., selectable marker). For example, in certain embodiments, methods are provided wherein a cell comprises a zinc finger protein whose expression is operably linked to a transcription control element responsive to a known molecular target, preferably a component of a cellular process such as a biochemical pathway or signal transduction pathway. Thus, under conditions in which the signal transduction pathway is active, the zinc finger protein is expressed. The cell also preferably comprises a polynucleotide encoding a reporter (e.g., a fluorescent protein, such as green fluorescent protein, a luciferase, a beta-galactosidase, a beta-glucuronidase, a beta-lactamase, a peroxidase such as horseradish peroxidase, an alkaline phosphatase, CAT, etc.). A subset of reporter molecules includes selectable markers (e.g., drug resistance, thymidine kinase, etc.). The reporter or selectable marker can be operably linked to transcriptional control elements that are modulated by the zinc finger protein (or fusion containing the zinc finger protein). Accordingly, when a test compound is administered to the cell, the ability of the compound to interact with a target (e.g., a component of a signal transduction pathway) to modulate production of the ZFP will be reflected in the amount of reporter and/or selectable marker produced. In certain embodiments, the zinc finger protein (or fusion) represses expression of the reporter and/or selectable marker. In these cases, if the compound interacts with its target in such a way as to block a signal transduction pathway, production of a reporter whose expression is controlled by the ZFP is increased. The cells may also comprise more than one reporter and/or selectable marker.
In additional embodiments, cells which overexpress a molecular target are provided, wherein overexpression is mediated by the action of an exogenous zinc finger protein. Similarly, cells which underexpress a molecular target, wherein underexpression is mediated by the action of an exogenous zinc finger protein, are provided. Methods of making and using such cells are also provided.
In still further embodiments, methods are provided that involve using one or more cellular components isolated from a cell containing an exogenous zinc finger protein. In preferred embodiments, the cellular component is cell membranes which are preferably isolated from cells that overexpress a molecular target (e.g., receptor) by virtue of the activity of the zinc finger protein (or fusion containing same). The isolated membranes can be used, for example, for binding studies (e.g., using radiolabeled ligands).
In another aspect, cells comprising any of the zinc finger proteins described herein are provided. In preferred embodiments, a cell that expresses a molecular target is provided. A cell of this sort exhibits a property that is responsive to the cell being contacted with a compound that interacts with the molecular target. The cell contains an exogenous zinc finger protein that modulates the production of a protein in the cell, preferably the molecular target.
In yet another aspect, kits for screening a compound of interest comprising any of the cells, proteins, polynucleotides and the like described herein are provided. In preferred embodiments, the kit further comprises ancillary reagents, instructions and other materials designed to carry out of the methods of screening described herein.
These and other embodiments will be readily apparent to the skilled artisan in view of the disclosure herein.